The Bean Beetle Microbiome Project (www.beanbeetles.org) provides a set of class-tested protocols that permit undergraduates to conduct original research. Using a course-based undergraduate research experience (CURE) approach, students explore the bacterial microbiome communities found in the digestive tract of bean beetles, Callosobruchus maculatus. Female bean beetles attach individual fertilized eggs to the surface of the host bean in which their offspring feed, develop, and pupate. We have developed a simple and reliable method for students to obtain bean beetle eggs to conduct bacterial microbiome studies. Individual fertilized adult female bean beetles were introduced to a small number (4-5) of sterile 5 or 6mm glass beads in a 35mm sterile Petri dish. Females readily laid eggs on these glass beads. The collection of glass beads with eggs (at least 5 eggs total) from one female provided enough material to either extract the bacterial microbiome for culturing or to perform DNA extraction for diversity assay sequencing of the V4 region of the 16S rRNA bacterial gene. Eggs laid on glass beads cannot develop beyond the embryonic and first instar larval stage since they cannot burrow into a substrate, and they are prevented from feeding. We surface sterilized the beads and eggs in the same manner as adult beetles (Cole et al., 2018) by placing the beads in a cell strainer cup before dipping through a sequence of bleach, sterile water, ethanol, and then sterile water. A sterile 2mL microfuge tube containing 4-5 glass beads with saline (for culture plating) or the first extraction buffer (for DNA extraction) placed in a pulsing vortex machine for 60 seconds will crush the eggs and strip the eggs from the beads. This technique has permitted our students to successfully perform studies comparing the bacterial microbiomes of virgin adult bean beetles to eggs and embryos
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